Von Willebrand factor (vWF) is a glycoprotein circulating in plasma as a series of multimers ranging in size from about 500 to 20,000 kD. Multimeric forms of vWF are composed of 250 kD polypeptide subunits linked together by disulfide bonds. vWF mediates the initial platelet adhesion to the sub-endothelium of the damaged vessel wall, only the larger multimers also exhibiting hemostatic activity. It is assumed that endothelial cells secret large polymeric forms of vWF and that those forms of vWF which have a low molecular weight (low molecular weight vWF) have arisen from proteolytic cleavage. The multimers having large molecular masses are stored in the Weibel-Pallade bodies of the endothelial cells and liberated upon stimulation.
vWF is synthesized by endothelial cells and megakaryocytes as prepro-vWF that consists to a large extent of repeated domains. Upon cleavage of the signal peptide pro-vWF dimerizes through disulfide linkages at its C-terminal region. The dimers serve as protomers for multimerization which is governed by disulfide linkages between the free end termini. The assembly to multimers is followed by the protelytic removal of the propeptide (Leyte et al., Biochem. J. 274 (1991), 257–261).
The full length of cDNA of vWF was cloned; the propolypeptide corresponds to amino acid residues 23 to 764 of the full length prepro-vWF (Eikenboom et al (1995) Haemophilia 1, 77–90).
The propeptide of vWF (pp-vWF) was shown to be identical to the von Willebrand antigen II, the second identified antigen that is deficient in the plasma and platelets of patients with severe von Willebrand disease (vWD). pp-vWF is specifically localized in platelets since plasma contains less than 5% of total propeptide vWF in blood, assuming the platelet count is 3×108 per ml. As already known, pp-vWF is released from platelets upon activation by various agonists. The pp-vWF is a glycoprotein not only because it reacts with periodic acid Schiff's reagent but also because it binds to lentil lectin. pp-vWF binds specifically to native type I collagen, but does not bind to heat-denatured collagen. It was shown that the affinity between pp-vWF and type I collagen was quite high so that the binding—which does not require any divalent cation and is not affected by addition of a peptide that contains sequence of arginine-glycine-aspartic acid (that is known to inhibit many cell attachment processes)—rapidly reached equilibrium.
The physiological role of pp-vWF is postulated to lie in the government of the assembly of vWF multimers, either before or after the cleavage from pro-vWF molecules. (Takagi et al., JBC 264 (18) (1989), 10425–10430).
pp-vWF was also shown to inhibit the platelet collagen interaction action (Takagi et al., JBC 264(11) (1989), 6017–6020).
In Isobe et al. (JBC 272 (13) (1997), 6447–6453) the role of pp-vWF as a novel physiological ligand and an adhesion substrate for α4β1 integrin-expressing leukemia cells was investigated. It was found that pp-vWF plays an important role in the mechanism underlying the melanoma metastasis as well as vascular inflammation.
Although pharmaceutical preparations containing mature vWF are known (see e.g. U.S. Pat. No. 5,571,784) the pharmaceutical usage of pp-vWF or the pro-form vWF have not been described or suggested in the prior art. According to the U.S. Pat. No. 5,571,784 vWF does not impair the systemic anticoagulatory effect of the anticoagulant hirudin as measured by the aPTT, rather it decreases the bleeding side effects of anticoagulant therapy. vWF is therefore proposed as a pseudo-antidote in assocation with hemorrhages which are produced by administering antithrombotic and/or fibrinolytic agents.
From Blann et al. (Eur. J. Vasc. Surg. 8 (1994), 10–15) it is also known that vWF levels are increased with risk factors for atherosclerosis and in patients with diffuse arterial disesase. The level of vWF is also thought to be a measure of endothelial 4>integrin-expressing leukemia cells was investigated. It was found that pp-vWF plays an important role in the mechanism underlying the melanoma metastasis as well as vascular inflammation.